Ovulation is triggered by neural circuits in the brain, which senses a rising tide of estradiol (E2) and-at the right time-generate a surge of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), causing ovulation. However, the cellular and molecular pathways in the brain that orchestrate this phenomenon are only partially understood. In rodent species, the anteroventral periventricular nucleus (AVPV) comprises part of the circuitry necessary to produce the GnRH/LH surge;however, until recently, the phenotype of the neurons within the AVPV that serve this function were a mystery. Within the past 3 years, it has become widely accepted that a product of the Kiss1 gene, kisspeptin, provides an important-perhaps essential-signal to GnRH neurons. The overall goal of this proposal is to identify the role that Kiss1 neurons in the AVPV play in the generation of the GnRH/LH surge and to reveal the neural, hormonal, and molecular pathways involved in that process. The first specific aim is to determine whether Kiss1 neurons in the AVPV and kispeptin produced by those particular neurons are essential for generating the GnRH/LH surge and to delineate the biophysical properties of those neurons as a function of the surge. Progesterone receptor (PR) signaling is an essential component of the surge mechanism, but the cellular and molecular basis of PR's action in the brain as it relates to the surge is not known. The second specific aim is designed to determine the functional significance of PR in Kiss1 neurons of the AVPV. The third specific aim is to identify the neural afferents and signaling pathways that control Kiss1 neurons in the AVPV and to evaluate their physiological significance in the context of GnRH/LH secretion. The experimental approach combines more traditional methodologies, such as in situ hybridization, immunohistochemistry, and hormone manipulations and measurements, together with innovative gene-targeting strategies. These include methods to 1) identify Kiss1 neurons with GFP and tdTomato for recording in slice preparations;2) ablate specific neurons through the use of selective diphtheria toxin receptor expression;3) map the afferent inputs to Kiss1 neurons with retrograde tracing by introducing a fluorescent-tagged pseudorabies virus into Kiss1 neurons;4) knock-down and knock-in specific genes in Kiss1 neurons with the use of a lentivirus delivery system;and 5) fingerprint the transcriptome of Kiss1 neurons by harvesting individual cells and employing a new "ribotagging" methodology. The studies described in this proposal utilize a multi-disciplinary approach to advance our understanding of a critical element in the female reproductive life cycle-the neuroendocrine mechanism that governs ovulation. PUBLIC HEALTH RELEVANCE: RELEVANCE Elucidating how the brain triggers the neuroendocrine events that lead to ovulation could provide the intellectual platform for understanding certain disorders of female reproduction, including delayed or precocious puberty and menstrual cycle dysfunction-and perhaps guide us toward improved therapies for their treatment. This knowledge could also serve as the basis for the development of newer and better strategies for hormonal contraception and infertility treatment-thus, offering hope and freedom of choice to women and their loved ones who are engaged in family planning.